Novel dna synthase

ABSTRACT

The object of the invention is to provide proteins that have both DNA primase activity and DNA polymerase activity. This subject is solved by a protein (p41) that has an amino acid sequence shown in SEQ ID NO: 1. This is for the first time that proteins that have both DNA primase activity and DNA polymerase activity were found. A protein (p46) that has amino acid sequence shown in SEQ ID NO: 2 forms a complex with p41, and enforces DNA synthesis activity that is independent and/or dependent from primer of p41.

TECHNICAL FIELD

[0001] The present invention relates to a new protein that is useful asa genetic engineering reagent and a method to produce the protein.

BACKGROUND ART

[0002] A DNA polymerase that synthesizes DNA chain having a sequencethat is complementary to base sequence of the template DNA is usedcommonly as an essential reagent for genetic engineering experimentslike PCR (Polymerase Chain Reaction), a base determination of DNA, asite-specific mutagenesis, and so on. Contribution of the enzyme to theprogress of molecular medicine, molecular biology, and biochemistry isenormous.

[0003] To examine the enzyme called a DNA polymerase closely,biochemical character of each enzyme is different, and various DNApolymerases are sold in the market until now. Each enzyme has differentcharacteristics like thermo stability, synthesized chain elongationability, ability to proofread wrong base in synthesizing, preference ofa template DNA, and they are selected according to the object of theexperiment.

[0004] However, these enzymes are not sufficient to satisfy all theobjects of experiments. More suitable new DNA polymerase is desired tobe developed to each object. Also, as the basic characteristic of DNApolymerase, short chains of nucleotides, a primer, are essential inorder to start DNA synthesis reaction, so the PCR needs a pair ofsite-specific primers for amplification area, and, it is necessary toprepare the primer that amplifies target area and the primer needs to beadded to a reaction mixture for each experiment.

DISCLOSURE OF THE INVENTION

[0005] The object of the present invention is to identify gene for newDNA synthetase and to provide the new DNA synthetase having a newbiochemical characteristic, as a reagent for genetic engineering.

[0006] The present invention relates to proteins that show DNA primaseactivity and DNA polymerase activity.

[0007] The “DNA primase activity” described in the present specificationmeans an ability to synthesize a DNA chain with substrate describedbelow by depending on a template DNA chain when a DNA chain that couldbe a template and deoxynucleotide triphosphate that could be substrateis present. In the same way, the “DNA polymerase activity” means anability to synthesize a DNA chain from 3′ terminal of a primer withsubstrate described below by depending on a template DNA chain when atemplate-primer, template DNA chain that is bound with its complementaryoligodeoxynucleotide (primer), and deoxynucleotide triphosphate thatcould be substrate is present. This is the first time that a proteinhaving both DNA primase activity and DNA polymerase activity was found.

[0008] This protein could be derived from eukaryotes including mammals,or prokaryotes including archaebacteria, eubacteria. In the preferredembodiment, this protein derives from archaebacteria, and having thermostability. In the present invention, protein that has “thermo stability”means, the protein could preserve the activitiesunder temperature of 50°C. or more.

[0009] In a one embodiment, the protein includes amino acid sequenceshown in SEQ ID NO: 1. In another embodiment, the protein may includesamino acid sequence wherein one or several amino acids are deleted,replaced or added in amino acid sequence shown in SEQ ID NO: 1.

[0010] In the present specification, these proteins are sometimes called“protein 1”.

[0011] The present invention also relates to a protein that couldenforces the DNA primase activity and/or the DNA polymerase activity byforming a complex with the protein that is described above. This proteincould be derived from eukaryotes including mammals, or prokaryotesincluding archaebacteria, eubacteria. In case of the preferredembodiment, this protein derives from an archaebacteria, and havingthermo stability. In one embodiment, the protein includes amino acidsequence shown in SEQ ID NO: 2. The protein may includes amino acidsequence wherein one or several amino acids are deleted, replaced oradded in amino acid sequence shown in SEQ ID NO: 2.

[0012] In the present specification, these proteins are sometimes called“protein 2”.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a photograph showing DNA primase activity of p41. Thisis an autoradiography of the product separated by electrophoreting withalkaline agarose gel after a reaction according to a method described inExample 5. When the template DNA existed, p41 synthesized 0.4 to 0.6kilo base pairs of DNA chain. When DNA polymerase I was added together,longer chain product was detected.

[0014]FIG. 2 shows a photograph of primase activity of p41. Anautoradiography of the product separated by electrophoreting with 10%polyacrylamide gel including 8M of urea after the reaction according toa method described in Example 5. 30-chain-length primers labelled withradiation were elongated by the p41 as well as DNA polymerases I and II.The synthesized chains were becoming longer as the time elapses.

[0015]FIG. 3 shows a photograph of primase activity of p41. To analyzereacted products in FIG. 2 more thoroughly, the same reaction mixturewas analyzed by electrophoreting with 1% alkaline agarose gel.

[0016]FIG. 4 shows a photograph of p41-p46 complex. A purified p46 waseluted to non-absorption area of a cation exchange column chromatography(lane 3). p41 was absorbed and eluted with about 0.6 M of NaCl (lane 1).Mixing both proteins and set the mixture to the column, both of themwere eluted by the same concentration of NaCl (lane 2), which supportsthat the both proteins would form stable complex.

[0017]FIG. 5 shows a measurement of affinity to DNA. The purifiedp41-p46 complex was used to three types of DNA to see avidities. Ratioof protein to DNA was 0, 1, 3, 5, starting from lane 1 to 4. All threetypes of DNA showed band shift according to the amount of protein.

[0018]FIG. 6 shows a progress of DNA synthesis reaction by p46. DNAsynthesis reaction (DNA primase assay) using p41 alone or p41-p46complex was executed and studied by electrophoreting with alkalineagarose gel. Even under the condition that the product would not find bythe reaction of the p41 alone (lane 1), extremely strong signal of theproduct was detected from complex (lane 2 and 3). When the quadruplereaction mixture of p41 alone was electrophoreted, a product wasdetected.

[0019]FIG. 7 shows progress of DNA synthesis reaction by p46. Using p41alone or p41-p46 complex, DNA synthesis reaction (DNA polymerase assay)was executed. The reaction mixture was electrophoreted with alkalineagarose gel after 5 minutes and 10 minutes of reaction and studied.Compared to the reaction of p41 alone, complex had shorter chain butextremely strong signal is detected.

BEST MODE OF CARRYING OUT THE INVENTION

[0020] The inventors of the present invention set their goal to isolateDNA synthetase that has excellent characteristic as a geneticengineering reagent, especially thermo stability, and screenedhyperthermophilic archaebacteria. In the genome sequence of Pyrococcusfuriosus, which was isolated and identified as hyperthermophilicarchaebacteria, there are genes that may code similar sequence toeukaryote DNA primase. Also, investigating this gene area, another genewas found just next to this area, and it was predicted that these geneareas form operon. Accordingly, the inventers of the present inventioncloned these genes (SEQ ID NO: 3 and 4), and produced and purified thecoded proteins, and identified biochemical characteristic of theseproteins. The purified proteins are named p41 (SEQ ID NO: 1) and p46(SEQ ID NO: 2).

[0021] DNA primer synthesizing (primase) activity was detected from p41in vitro reaction. Further, contrary to what had expected, p41 showedstrong primer extending activity that is as strong as a conventional DNApolymerase. These activities are very stable under heat and have a greatpotential to be utilized as an amplification enzyme for gene. It isespecially notable that the protein has the activity to synthesize DNAchain without primer. In order to amplify the target gene area, thecurrent PCR technique needs to make a specific primer for each area, andadd them to the reaction mixture. The enzyme of the present inventionhas a DNA-synthesizing activity and does not need a primer, so the newgene amplification technique could be provided. The inventors also foundout that a protein derived from hyperthermophilic archaebacteriaAeropyrum pernix has same characteristic and shows the DNA primaseactivity and the DNA polymerase activity.

[0022] No DNA synthesis reaction was detected by p46 alone, but it wasfound that p46 formed stable complex with p41, and contributes toincrease affinity to DNA. DNA synthesizing activity that is independentfrom primer in p41-p46 complex showed much higher rate compared to p41alone. Also, p41-p46 complex can synthesize RNA primer even though it isnot so effective.

[0023] Therefore, the present invention is a method to polymerizedeoxynucleotide triphosphate and also relates to,

[0024] 1) provide DNA chain as template DNA and deoxynucleotidetriphosphate as substrate,

[0025] 2) add protein 1 and polymerize deoxynucleotide triphosphate. Theprotein 2 can be added in this polymerization method besides theprotein 1. The protein 2 enforces the DNA primase activity of theprotein 1.

[0026] The present invention is also a method to polymerizedeoxynucleotide triphosphate and also relates to,

[0027] 1) provide DNA chain as template DNA and deoxynucleotidetriphosphate as substrate,

[0028] 2) add complementary oligodeoxynuclotide (primer) to DNA chain astemplate DNA and form complex with the DNA chain,

[0029] 3) add protein 1 and polymerize deoxynucleotide triphosphate.

[0030] The protein 2 can be added in this polymerization method besidesthe protein 1. The protein 2 enforces the DNA polymerase activity of theprotein 1.

[0031] As mentioned above, the inventors has identified the unkown twoproteins,the former that has both the DNA primase activity and the DNApolymerase activity, and the latter that enforces the DNA polymeraseactivity of the former one. These proteins are used for a new geneticengineering method which utilizes the DNA synthesis reaction.

[0032] The protein 1 or 2 according to the present invention aresynthesized recombinant DNA method described below:

[0033] 1) prepare a base sequence that codes the protein 1 or 2,

[0034] 2) insert the base sequence to an expression vector,

[0035] 3) transform host cell by the vector,

[0036] 4) cultivate the transformant, and

[0037] 5) isolate desired protein from the culture.

[0038] SEQ ID NO: 3 is shown as an example for the base sequence thatcodes the protein 1. It is well known that many other base sequences canbe used to code protein 1 by degeneration of genetic codes.

[0039] SEQ ID NO: 4 is shown as an example for the base sequence thatcodes the protein 2. It is well known that many other base sequences canbe used to code protein 2 by degeneration of genetic codes.

[0040] To make purification of the expressed protein easier, or forother purposes, it is possible to express the protein of the presentinvention as the fusion protein of the protein in the present inventionand other proteins or peptides. In that case, base sequence that codesthe proteins of the present invention needs binding method with basesequences that code other proteins or peptides by appropriate.

[0041] The present invention relates to a vector, especially a plasmid,a cosmid, a virus, a bacteriophage, and other vectors that are generallyused for genetic engineering. By using methods that are well known tothose skilled in the art, the varieties of plasmids and vectors arebuild. (For example, refer to the technique described in Sambrook,Molecular Cloning A Laboratory Manual, Cold spring Harbor Laboratory(1989) and Ausbel, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y. (1994)). According tothe present invention, the plasmids and vectors that are preferably usedinclude the plasmids and vectors that are well known to those skilled inthe art.

[0042] In the preferred embodiments, a DNA sequence that codes a proteinof the present invention inside vector binds in active state with thecontrol sequence that is needed in order to express genes in procaryoticor eukaryotic cell.

[0043] The word “control sequence” means a control DNA sequence that isneeded to express the code sequence that binds with. Characteristic ofsuch a control sequence changes by a host. The control sequence inprokaryote cell generally includes a promoter, a ribosome binding siteand terminator. The control sequence in eukaryotic cell includes apromoter, a terminator, and in some cases, a transactivator, or atranscription factors. The word “control sequence” intends to includeall the elements that are needed to express its presence by the smallestunit. Also it is possible for the “control sequence” to include someother useful elements.

[0044] The word “binds in active state” means each of the elements is ina position that is possible to work in the intended method. The controlsequence that “binds in active state” to the control sequence is boundby a method that can achieve the expression of codes, and the conditionis in the same range as the control sequence. In case the controlsequence is a promoter, fact that the double-stranded DNA is favored iswell known to those skilled in the art.

[0045] Therefore, a vector that is used for the present invention is anexpression vector. The “expression vector” is a construction thattransforms a selected host cell, and expresses the code sequence insidethe selected host cell. The expression vector may be, for example, acloning vector, a binary vector, or an integrating vector. Theexpression favorably includes a transcription of the nucleic acidmolecule to mRNA which is possible to translate.

[0046] A controlling element that ensures the expression inside theprokaryote cells and/or eukaryote cells is well known to those skilledin the art. In the case of eukaryote cells, the controlling elementusually includes a promoter that ensures starting of the normaltranscription and poly (A) signal that ensures the end of transcriptionand stability of transcript. The promoters that are used in general area polyubiquitin promoter and an actin promoter. Further, the controllingelement may include transcription or translation enhancer.

[0047] Examples of the possiblecontrolling element that enables theexpression in prokaryote cells are PL, lac, trp or tac promoters for E.Coli. In the eukaryote cells, examples of the possible controllingelements that enable the expression includes AOX1 or GAL1 promoters foryeast, and CMV-, SV40-, RSV-promoters (Rous Sarcoma virus) are CMVenhancer, SV40 enhancer or globin intron for mammals and other animalcells. The expression vectors that are appropriate and well known tothose skilled in the art are the expression vector pcDV1 (Pharmacia) byOkayama-Berg, pCDM8, pRc/CMV, pcDNA1, pcDNA3 (In-vitrogen), pSPORT1(GIBCO BRL), and so on.

[0048] In the preferred embodiment, the vector in the present inventionthat is described above includes selectable markers.

[0049] Further, the present invention relates to a host cell thatincludes a vector described above. Sequence of nucleic acid is foreignto the host cell.

[0050] The word “foreign” means the nucleic acid molecule isheterologous (it means that the nucleic acid molecule derives fromdifferent cell or a creature having different genetic background) to thehost cell, or the nucleic acid molecule is homologus to the host cell,but in a different genetic environment from the counterpart that isnaturally present in the nucleic acid molecule. That means, if thenucleic acid molecule is homologus relating to the host cell, thenucleic acid molecule in host cell is not in natural gene position. Inthis case, the nucleic acid molecule is in control under the promoter ofitself, or can be under the control of heterologous promoter. The vectoror the promoter of the present invention inside host cell can beintroduced into genome of the host cell or kept outside chromosomes.

[0051] Therefore, the present invention relates to the host cell thatincludes a vector or genes of the present invention. The host cell canbe prokaryote or eucaryote cell, for example eubacteria, archaebacteria,insects, fungi, plants, or animal cells etc. Favorable fungi cells are,for example, cell of genus Saccharomycess, especially Saccharomycesscerevisiae.

[0052] The word “prokaryote” intends all the bacteria that can betransformed or transfected by DNA or RNA to express the protein of thepresent invention. Examples of prokaryotic host cells are gram positiveand negative bacteria like E. coli, S. typhimurium, Serratia marcescens,or Bacillus subtilis, and may include archaebacteria Methanococcusmaripaludis, or Haloferax volvanii.

[0053] The word “eukaryote” intends to include yeast, higher plants,insects, and favorably, cells of mammals. For the host cells used forrecombination producing method, the protein that is coded by thepolynucleotides of the present invention may be glycosylated or may benot. The protein of the present invention may or may not have the firstamino acid residue.

[0054] Using one of the techniques well known to those skilled in theart, genes of the present invention is transformed, or transfected.Further, production method for fused, or functionally bound gene andalso a method to express the gene inside for example, mammals orbacteria is well known to those skilled in the art (Sambrook, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989).

[0055] The recombinant Escherichia coli bacteria that is used to producenew protein described in the present invention is named and expressed asEscherichia coli BL21(DE3)RIL/pPFPR41 and Eschericia coliBL21(DE3)RIL/pPFPR46. These Escherichia coli bacteria are deposited tothe national institute of advanced industrial science and technology asaccession number FERM BP-7650 and FERM BP-7651.

[0056] Embodiments of the present invention will be explained in detailbelow. It should be noted that the present invention is not limited tothe below-explained embodiments.

EXAMPLES Example 1

[0057] Preparing Genomic DNA of Pyrococcus furiosus

[0058]P. furiosus DSM3638 was obtained from Deutsche Sammlung vonMikroorganismen und Zelkulturen GmbH and cultured by the methoddFescribed in the document (Nucleic Acids Research, Vol. 21 p259-265).1.2 g of cell body was extracted from 500 ml of culture solution. Theextracted cell body was suspended with 10 ml buffer L (10 mM Tris-HCl(pH8.0), 1 mM EDTA, 100 mM NaCl), and added 1 ml of 10% SDS. After thesuspension was stirred, 50 μl of proteinase K (20 mg/ml) was added, andthe suspensionwas left still for 60 minutes under temperature of 55° C.Then the reaction mixture went under the process of phenol extraction,phenol extraction/chloroform extraction, and chloroform extractionsequentially. DNA was then insolubilized by adding ethanol. CollectedDNA was dissolved in 1 ml of TE (10 mM Tris-HCl, pH8.0, 1 mM EDTA), andreacted with 0.75 mg of RNase A for 60 minutes under temperature of 37°C. Then the reaction mixture goes under the process of phenolextraction, phenol extraction/chloroform extraction, and chloroformextraction sequentially. DNA was collected by the ethanol precipitation.DNA was then insolubilized by adding ethanol. 0.75 mg of DNA iscollected.

Example 2

[0059] Cloning of the gene that codes Pyrococcus furiosus p41 and p46

[0060] To clone the target gene from the genome DNA of the Pyrococcusfuriosus, an adequate primer was synthesized. As the gene amplifier ofp41, 5′CATATGCTGATGAGGGAAGTGACAAAGGAG-3′5′CTCGAGCCTTTATTCATATTCCAAGGACTCT-3′

[0061] were used as the forward and reverse primer. For amplifying thegene p46, 5′-CACGACCATGGTAGACCCATTTAGTGAG-3′5′-CACGGTCGACTCATTACTGTAGAATTCGCT-3′

[0062] were used as the primers. PCR was used under normal composition,the time and temperature of one cycle was 30 seconds with 95° C., 30seconds with 55° C., 30 seconds with 72° C. for 30 cycles. The amplifiedDNA chain was inserted to pT7 blue vector (Novagen, Inc) and recombinantplasmid was isolated. The primers were provided with a recognitionsequence that recognizes restriction enzymes NdeI, XhoI (p41), or NdeI,SalI (p46) respectively. By digesting DNA with those enzymes, only thetarget part was cut. The target part was integrated to expression vectorpET type with ATG in the NdeI sequence of the target part as aninitiation codon for translation. The p41 and p46 genes (SEQ ID NO: 3and 4) were inserted to pET28a′ (pET28a originally has kanamycinresistance gene as a selectable marker but it is replaced withampicillin resistant marker for convenience) and pET21a. The plasmidswere named pPFPR41 and pPFPR46. By constructing such plasmids, p41 wasproduced in a form with a tag having six histidine connected toN-terminal, and p46 was produced in a form that start with the originalinitiation codon ATG when they were translated.

Example 3

[0063] Production and Purification of p41 Protein

[0064] The plasmid pPFPR41 was inserted to Escherichia coliBL21(DE3)RIL, cultured the transformant that was obtained, and producedthe target protein.

[0065] The Escherichia coli BL21(DE3)RIL/pPFPR41 was cultured in 500 mlof LB culture (tryptone 10 g/l, yeast extract 5 g/l, NaCl 5 g/l, pH7.2)with 100 μg/ml concentration of ampicillin and 20 μg/ml concentration ofchloramphenicol. When turbidity of the culture became 0.4A₆₀₀, inducer,isopropyl-β-D-thiogalactoside (IPTG), was added and then cultured forfive hours. After collecting cell bodies, the cell bodies were suspendedwith 40 ml buffer A (50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 2 mMβ-mercaptoethanol) and 1 ml of phenylmethylsulfonyl fluoride (PMSF). Thesuspension was put to ultrasonic disintegrator. Supernatant of theextraction was collected after 20 minutes of centrifugal separation by16,000 rpm, and boiled for 15 minutes with 80° C. Almost all of theprotein derived from Escherichia coli was denatured and insolubilized.Then the supernatant was collected by using the centrifugal separationand poured onto the metal resin (Co²⁺) chelating column (TALON, ClontechLaboratories Inc.). The proteins that were not conjugated were washedand removed by the buffer A including 10 mM of imidazole. Then theconnected proteins with histidine tag on it were eluted by make theconcentration rate of the imidazole higher to 100 mM. This fraction waspoured onto a cation-exchange column (HiTrap SP, Amersham PharmaciaBiotech) and chromatography was carried out with automatic liquidchromatography system (AKTA explorer, pharmacia). It was developed from0.1 to 0.8 M of NaCl according to linear NaCl gradient slope. The targetactivity was found at 0.5 to 0.7 M NaCl. The fraction that showsactivity was collected and dialyzed with 2 l of the buffer A. Then thedialyzed fraction was purified and sampled. About 1 mg of enzyme wasextracted from 1 liter of culture.

Example 4

[0066] Production and Purification of p46 Protein

[0067] The plasmid pPFPR46 was inserted to Escherichia coliBL21(DE3)RIL, cultured the transformant that was obtained, and producedthe target protein.

[0068] The Escherichia coli BL21(DE3)RIL/pPFPR46 was cultured in thesame culture as described in the Example 3. After collecting cellbodies, these cell bodies were disintegrated and unrefined extract wascollected by centrifugal separation in the method described in theExample 3. Then the extraction was boiled for 15 minutes with 80° C.,and only the thermophylactic proteins were collected as the supernatantof the centrifugation. Polyethylenimine (Polymin P) and NaCl were addedto this solution for each concentration 0.2% (weight/capacity) and 0.3M.The solution was stirred with ice for 30 minutes. Insolubilized nucleicacid was removed by the centrifugal separation, and aluminum sulfate wasadded to saturate for 80%. As the result, the proteins wereprecipitated.

[0069] Precipitate were then dissolved to buffer C (50 mM Tris-Cl,pH8.0, 0.3 M NaCl, 1 mM β-mercaptoethanol) and poured onto theanion-exchange column (HiTrapQ, Amersham Pharmacia Biotech) afterequilibrating with same buffer C. It was developed from 0.1 to 1 M ofNaCl according to linear NaCl gradient slope. The target activity wasfound at 0.15 to 0.25 M NaCl. The fraction that shows activity wascollected for 10 ml and poured into heparin affinity column (HiTrapHeparin, Pharmacia Biotech). It was developed from 0.05 to 0.8 M of NaClaccording to linear NaCl gradient slope. The target protein was found at0.3 to 0.5 M NaCl. About 6.6 mg of enzyme was extracted from 1 liter ofculture.

Example 5

[0070] Detection of DNA Primase Activity

[0071] To detect the primase activity, the occurrence of the DNAsynthesizing was detected by tracing radio activity, by using M13 PhageDNA (single-stranded DNA) as a template and adding ³²P-labelled dATP asa part of substrate. Following were added to 50 mM Tris-HCl, pH8.1, 10mM MgCl₂, 1 mM β-mercaptoethanol solution with a concentration of: 0.05μg/μl of M13DNA, 100 μM of each dCTP, dGTP, dTTP, 10 μM of [α32p] dATP,and 0.7 μM of p41 protein or DNA polymerase I for 2.5 units. 20 μg ofthis reaction mixture was warmed for 20 minutes in 70° C., and separatedby whether electrophoreting with 10% polyacrylamide gel including 8M ofurea or electrophoreting with 1% alkaline agarose gel. FIG. 1 showsresult of electrophoreting with alkaline agarose gel. P41 proteinsynthesized 0.4 to 0.6 kbp of DNA chain when DNA template was present(lane 3). DNA polymerase I did not synthesize under this conditionbecause there were no primers present (lane 4). When pol I was added top41, synthesizing chain became longer (lane 5).

Example 6

[0072] Detection of DNA Polymerase Activity

[0073] To detect the polymerase activity, M13 Phage DNA (single-strandedDNA) was used as a template, and annealed with a primer that was 30chains long and labelled with ³²P at the 5′ terminal. It was thenreacted with enzyme sample with dNTP as a substrate. Following wereadded to 50 mM Tris-HCl, pH8.1, 10 mM MgCl₂, 1 mM β-mercaptoethanolsolution (20 μl), 0.5 μg of M13DNA that was annealed with the labeledprimer, 125 μM of each dCTP, dGTP, dTTP, 0.14 μM of p41 protein. Thereaction mixture was warmed for 5, 10, 20 minutes in 70° C., and takenout for 5 μl each, and added with 3 μl of stop solution (95% formamide,0.05% Bromophenol Blue, 0.05% Xylenecyanol). As the comparison, reactionmixture that was replaced p41 with 2.5 units of DNA polymerase I, or DNApolymerase II was experimented. The reaction mixture were separated byelectrophoreting with 10% polyacrylamide gel including 8M of urea orelectrophoreting with 1% alkaline agarose gel. Then autoradiography wastaken. As the result of electrophoreting with polyacrylamide gel, p41protein extended 30 chains long primer (FIG. 2), which was as same aspublicly known DNA polymerases I and II, so the reaction mixture waselectrophoreted with the alkaline agarose gel that enabled an analysisof long chain area (FIG. 3). At least under this condition, primerextending activity that was as equal as publicly known DNA polymeraseswere found.

Example 7

[0074] Forming p41-p46 Complex

[0075] To make sure the produced p41 andp46 interact, cation-exchangecolumn (HiTrap SP) was used to analyze with a single case, and mixedcase. As it was described in the Example 3, P41 protein was trapped bythe HiTrap SP column, but p46 passed through under the same condition.When the proteins were mixed and poured onto the column, p46 and p41were eluted together on a same absorbed fraction (FIG. 4). From thisresult, it was thought that both proteins formed very stable complex.

Example 8

[0076] Increase of DNA Binding Affinity by p46

[0077] The gel-shift assey was used to detect binding activity thatbinds enzyme to DNA. 49 chain-length oligodeoxynuclotide,5′-dAGCTACCATGCCTGCACGAATTAAGCAATTCGTAATCATGGTCATA GCT-3′ was labeledwith ³²P at 5′ terminal. Then the chain was used for gel-shift asseywith itself or annealed with 49 or 17 chain length complementaryoligodeoxynuclotide. With these three kinds of DNA, and the proteinswere mixed in variety of concentrations in gel-shift assey buffer (50 mMTris.Cl, pH8.0, 10 mM MgCl₂, 20 mM KCl, and 1 mM β-mercaptoethanol). Themixtures were left for 5 minutes under temperature of 55° C. andseparated by electrophoreting with 1% agarose gel. The gel was processedby 0.1×TAE buffer (4 mM Tris-Acetate, pH8.0, 0.1 mM EDTA) andelectrophoreted with same buffer. Autoradiography after theelectrophoration was shown in FIG. 5. p41-p46 complex bound to all typesof DNA and shifted the band as the complex. It was not shown on thediagram but under this condition, p41, p46 alone did not show binding(band shifts were not found).

Example 9

[0078] Increase of Efficiency in DNA Synthesize by p46

[0079] According to the method described in Example 5, effect of DNAsynthesis of p41-p46 complex was compared with the case when the eachprotein synthesizes the DNA alone.

[0080] Reacting condition was described as in Example 5 but as labelledcompound, [α32p] dCTP was used instead of [α32p] dATP. The separatedfraction by the cation exchange column chromatography described inExample 7 was used as sample of complex, but also in the case when theproteins that were purified alone respectively were added together atonce was experimented. FIG. 6 shows an analysis of the reacted productsby the electrophoration. When the reaction mixture of p41 alone (lane1), or p41-p46 complex (lane 2), and purified complex (lane 3) wereelectrophoreted for the same quantity, it was very clear that extremelystrong signal was detected from lane 2 and 3. p41 alone was difficult tofind a signal (lane 1), but when it was electrophoreted for a quadruple,it became much easier to find (lane 4). Even if the p41 became easier tofind, it still showed a weaker signal compared to products form thecomplex. Also, it was not shown on the diagram but p46 alone did notshow any DNA synthesis activity.

[0081] According to the method described in Example 6, effect of DNAsynthesis of p41-p46 complex that depended on the primer was comparedwith the case when the each protein synthesized the DNA with alone. AsFIG. 7 shows, compared with the reaction of p41 alone, the reaction ofp41-p46 complex produced much more products even though the length ofextending chain was short.

[0082] As described above, p46 enforces DNA synthesis activity of p41that is dependent or independent to the primer by forming complex.

1 9 1 347 PRT Pyrococcus furiosus 1 Met Leu Met Arg Glu Val Thr Lys GluGlu Arg Ser Glu Phe Tyr Ser 1 5 10 15 Lys Glu Trp Ser Ala Lys Lys IlePro Lys Phe Ile Val Asp Thr Leu 20 25 30 Glu Ser Arg Glu Phe Gly Phe AspHis Asn Gly Glu Gly Pro Ser Asp 35 40 45 Arg Lys Asn Gln Tyr Ser Asp IleArg Asp Leu Glu Asp Tyr Ile Arg 50 55 60 Ala Thr Ser Pro Tyr Ala Val TyrSer Ser Val Ala Phe Tyr Glu Asn 65 70 75 80 Pro Arg Glu Met Glu Gly TrpArg Gly Ala Glu Leu Val Phe Asp Ile 85 90 95 Asp Ala Lys Asp Leu Pro LeuLys Arg Cys Asn His Glu Pro Gly Thr 100 105 110 Val Cys Pro Ile Cys LeuGlu Asp Ala Lys Glu Leu Ala Lys Asp Thr 115 120 125 Leu Ile Ile Leu ArgGlu Glu Leu Gly Phe Glu Asn Ile His Val Val 130 135 140 Tyr Ser Gly ArgGly Tyr His Ile Arg Ile Leu Asp Glu Trp Ala Leu 145 150 155 160 Gln LeuAsp Ser Lys Ser Arg Glu Arg Ile Leu Ala Phe Ile Ser Ala 165 170 175 SerGlu Ile Glu Asn Val Glu Glu Phe Arg Arg Phe Leu Leu Glu Lys 180 185 190Arg Gly Trp Phe Val Leu Lys His Gly Tyr Pro Arg Val Phe Arg Leu 195 200205 Arg Leu Gly Tyr Phe Ile Leu Arg Val Asn Val Pro His Leu Leu Ser 210215 220 Ile Gly Ile Arg Arg Asn Ile Ala Lys Lys Ile Leu Asp His Lys Glu225 230 235 240 Glu Ile Tyr Glu Gly Phe Val Arg Lys Ala Ile Leu Ala SerPhe Pro 245 250 255 Glu Gly Val Gly Ile Glu Ser Met Ala Lys Leu Phe AlaLeu Ser Thr 260 265 270 Arg Phe Ser Lys Ala Tyr Phe Asp Gly Arg Val ThrVal Asp Ile Lys 275 280 285 Arg Ile Leu Arg Leu Pro Ser Thr Leu His SerLys Val Gly Leu Ile 290 295 300 Ala Thr Tyr Val Gly Thr Lys Glu Arg GluVal Met Lys Phe Asn Pro 305 310 315 320 Phe Arg His Ala Val Pro Lys PheArg Lys Lys Glu Val Arg Glu Ala 325 330 335 Tyr Lys Leu Trp Arg Glu SerLeu Glu Tyr Glu 340 345 2 396 PRT Pyrococcus furiosus 2 Met Leu Asp ProPhe Ser Glu Lys Ala Lys Glu Leu Leu Lys Glu Phe 1 5 10 15 Gly Ser MetAsn Glu Phe Leu Gln Ala Ile Pro Ser Leu Val Asp Ile 20 25 30 Glu Glu ValMet Asn Arg Leu Lys Phe Ala Lys Glu Ser Glu Ile Ser 35 40 45 Glu Asp IleLeu Asn Ile Glu Asp Ile Arg Asp Leu Ala Ser Phe Tyr 50 55 60 Ala Gln IleGly Ala Leu Ala Tyr Ser Pro Tyr Gly Leu Glu Leu Glu 65 70 75 80 Leu ValLys Lys Ala Asn Leu Arg Ile Tyr Thr Glu Arg Ile Arg Arg 85 90 95 Arg ArgLys Ile Arg Ser Asp Glu Ile Gly Ile Glu Val Lys Ile Ala 100 105 110 ValGlu Phe Pro Glu Asn Asp Ile Lys Thr Leu Glu Lys Val Tyr Gly 115 120 125Gly Leu Pro Glu Tyr Ile Val Ser Leu Arg Glu Phe Leu Asp Leu Val 130 135140 Pro Asp Glu Lys Leu Ser Ser Tyr Tyr Val Tyr Asp Gly Asn Val Tyr 145150 155 160 Leu Arg Lys Asp Asp Leu Leu Lys Val Trp Ser Lys Ala Phe GluArg 165 170 175 Asn Val Glu Lys Ala Val Asn Ile Ile Tyr Glu Ile Arg AspGlu Leu 180 185 190 Pro Glu Phe Tyr Arg Arg Leu Ala Gly Glu Ile Arg SerPhe Ala Glu 195 200 205 Lys Glu Phe Ser Asp Lys Phe Arg Glu Val Gln AlaGly Glu Leu Lys 210 215 220 His His Leu Phe Pro Pro Cys Val Lys Asn AlaLeu Arg Gly Val Pro 225 230 235 240 Gln Gly Met Arg Asn Tyr Ala Ile ThrVal Leu Leu Thr Ser Phe Leu 245 250 255 Ser Tyr Ala Arg Ile Cys Pro AsnPro Pro Arg Arg Asn Val Lys Ile 260 265 270 Arg Asp Cys Ile Lys Asp MetArg Val Ile Thr Glu Glu Ile Leu Pro 275 280 285 Ile Ile Ile Glu Ala GlyAsn Arg Cys Ser Pro Pro Leu Phe Glu Asp 290 295 300 Gln Pro Asn Glu IleLys Asn Ile Trp Tyr His Leu Gly Phe Gly Tyr 305 310 315 320 Thr Ala AsnPro Thr Leu Glu Asp Ser Gly Asn Ser Thr Trp Tyr Phe 325 330 335 Pro ProAsn Cys Asp Lys Ile Lys Ala Asn Ala Pro Gln Leu Cys Thr 340 345 350 ProAsp Lys His Cys Arg Tyr Ile Arg Asn Pro Leu Thr Tyr Tyr Leu 355 360 365Arg Arg Leu Tyr Leu Glu Glu Lys Arg Arg Ala Lys His Ala Asp Glu 370 375380 Gly Ser Asp Lys Gly Gly Lys Glu Arg Ile Leu Gln 385 390 395 3 1044DNA Pyrococcus furiosus exon (1)..(1041) 3 atg ctg atg agg gaa gtg acaaag gag gaa agg agc gaa ttc tac agt 48 Met Leu Met Arg Glu Val Thr LysGlu Glu Arg Ser Glu Phe Tyr Ser 1 5 10 15 aaa gaa tgg agt gca aag aaaata cca aag ttc ata gtg gac act cta 96 Lys Glu Trp Ser Ala Lys Lys IlePro Lys Phe Ile Val Asp Thr Leu 20 25 30 gaa agt aga gaa ttc ggc ttc gatcat aac ggg gaa ggt cca agt gac 144 Glu Ser Arg Glu Phe Gly Phe Asp HisAsn Gly Glu Gly Pro Ser Asp 35 40 45 agg aaa aat caa tat tct gac ata agagat tta gag gac tac att aga 192 Arg Lys Asn Gln Tyr Ser Asp Ile Arg AspLeu Glu Asp Tyr Ile Arg 50 55 60 gcc aca tcc ccc tac gca gta tat tca agtgtg gca ttt tat gaa aac 240 Ala Thr Ser Pro Tyr Ala Val Tyr Ser Ser ValAla Phe Tyr Glu Asn 65 70 75 80 ccc agg gag atg gaa ggg tgg aga gga gctgag tta gtt ttt gac att 288 Pro Arg Glu Met Glu Gly Trp Arg Gly Ala GluLeu Val Phe Asp Ile 85 90 95 gat gcc aag gat ctc ccc cta aag agg tgc aaccac gaa cct ggg aca 336 Asp Ala Lys Asp Leu Pro Leu Lys Arg Cys Asn HisGlu Pro Gly Thr 100 105 110 gtg tgt cca ata tgc ctt gaa gat gca aaa gagcta gct aaa gat act 384 Val Cys Pro Ile Cys Leu Glu Asp Ala Lys Glu LeuAla Lys Asp Thr 115 120 125 cta ata att ctc agg gaa gaa ctc ggc ttt gaaaat atc cat gta gtc 432 Leu Ile Ile Leu Arg Glu Glu Leu Gly Phe Glu AsnIle His Val Val 130 135 140 tac tcc gga aga gga tat cac ata aga atc ctagat gaa tgg gcc ctc 480 Tyr Ser Gly Arg Gly Tyr His Ile Arg Ile Leu AspGlu Trp Ala Leu 145 150 155 160 caa ttg gac tcc aaa agt aga gaa aga attctt gcc ttt att tca gct 528 Gln Leu Asp Ser Lys Ser Arg Glu Arg Ile LeuAla Phe Ile Ser Ala 165 170 175 agt gaa att gag aac gtt gaa gaa ttt agaaga ttt cta ctg gag aag 576 Ser Glu Ile Glu Asn Val Glu Glu Phe Arg ArgPhe Leu Leu Glu Lys 180 185 190 aga gga tgg ttt gtg tta aag cat ggc tacccg aga gta ttt agg ttg 624 Arg Gly Trp Phe Val Leu Lys His Gly Tyr ProArg Val Phe Arg Leu 195 200 205 aga ctg gga tac ttt att cta agg gtt aacgta cct cac ttg cta agc 672 Arg Leu Gly Tyr Phe Ile Leu Arg Val Asn ValPro His Leu Leu Ser 210 215 220 att gga ata aga aga aat att gca aag aaaatt cta gat cac aaa gaa 720 Ile Gly Ile Arg Arg Asn Ile Ala Lys Lys IleLeu Asp His Lys Glu 225 230 235 240 gaa ata tac gag gga ttt gta agg aaggca ata ttg gca tct ttt cca 768 Glu Ile Tyr Glu Gly Phe Val Arg Lys AlaIle Leu Ala Ser Phe Pro 245 250 255 gaa ggc gtg gga att gaa agc atg gctaag ctc ttt gcc cta tca act 816 Glu Gly Val Gly Ile Glu Ser Met Ala LysLeu Phe Ala Leu Ser Thr 260 265 270 aga ttt tca aag gcc tat ttt gat ggtagg gtt aca gtt gat ata aag 864 Arg Phe Ser Lys Ala Tyr Phe Asp Gly ArgVal Thr Val Asp Ile Lys 275 280 285 aga atc cta agg ttg ccc tca aca ctccat tcc aaa gtg ggc ctt ata 912 Arg Ile Leu Arg Leu Pro Ser Thr Leu HisSer Lys Val Gly Leu Ile 290 295 300 gca act tat gtt gga acc aag gag agagag gtc atg aag ttt aat cca 960 Ala Thr Tyr Val Gly Thr Lys Glu Arg GluVal Met Lys Phe Asn Pro 305 310 315 320 ttt aga cat gca gtg cca aag ttcagg aaa aaa gaa gtg cgc gaa gct 1008 Phe Arg His Ala Val Pro Lys Phe ArgLys Lys Glu Val Arg Glu Ala 325 330 335 tat aaa ctg tgg aga gag tcc ttggaa tat gaa taa 1044 Tyr Lys Leu Trp Arg Glu Ser Leu Glu Tyr Glu 340 3454 1191 DNA Pyrococcus furiosus exon (1)..(1188) 4 atg cta gac cca tttagt gag aag gcc aaa gaa cta cta aaa gaa ttc 48 Met Leu Asp Pro Phe SerGlu Lys Ala Lys Glu Leu Leu Lys Glu Phe 1 5 10 15 gga tca atg aat gaattc ctt caa gct atc ccc tct ctt gtg gat ata 96 Gly Ser Met Asn Glu PheLeu Gln Ala Ile Pro Ser Leu Val Asp Ile 20 25 30 gag gaa gtc atg aat aggtta aaa ttt gca aaa gaa tcc gaa atc tcc 144 Glu Glu Val Met Asn Arg LeuLys Phe Ala Lys Glu Ser Glu Ile Ser 35 40 45 gaa gat att ctg aat ata gaggat ata cga gat tta gca agc ttt tat 192 Glu Asp Ile Leu Asn Ile Glu AspIle Arg Asp Leu Ala Ser Phe Tyr 50 55 60 gcc caa ata gga gca tta gct tactcc cca tat gga ctg gaa ttg gaa 240 Ala Gln Ile Gly Ala Leu Ala Tyr SerPro Tyr Gly Leu Glu Leu Glu 65 70 75 80 cta gta aag aag gct aat ttg agaata tat aca gag aga atc cgc aga 288 Leu Val Lys Lys Ala Asn Leu Arg IleTyr Thr Glu Arg Ile Arg Arg 85 90 95 aga agg aaa ata agg agc gat gaa attgga att gaa gta aaa ata gca 336 Arg Arg Lys Ile Arg Ser Asp Glu Ile GlyIle Glu Val Lys Ile Ala 100 105 110 gtt gaa ttc cca gaa aac gac ata aaaaca ctt gaa aaa gtc tat ggt 384 Val Glu Phe Pro Glu Asn Asp Ile Lys ThrLeu Glu Lys Val Tyr Gly 115 120 125 ggc ctt cca gaa tac ata gtt tcc ctaagg gag ttt tta gat cta gtt 432 Gly Leu Pro Glu Tyr Ile Val Ser Leu ArgGlu Phe Leu Asp Leu Val 130 135 140 cca gat gaa aaa ctc tcc tct tat tacgtc tat gat ggg aat gtg tat 480 Pro Asp Glu Lys Leu Ser Ser Tyr Tyr ValTyr Asp Gly Asn Val Tyr 145 150 155 160 tta agg aag gat gac ctc tta aaagtg tgg agc aaa gct ttt gag aga 528 Leu Arg Lys Asp Asp Leu Leu Lys ValTrp Ser Lys Ala Phe Glu Arg 165 170 175 aac gtt gaa aag gcc gtg aat ataatt tac gaa ata agg gac gag ctt 576 Asn Val Glu Lys Ala Val Asn Ile IleTyr Glu Ile Arg Asp Glu Leu 180 185 190 cca gag ttt tat aga aga ctt gcagga gag ata aga tct ttt gcc gag 624 Pro Glu Phe Tyr Arg Arg Leu Ala GlyGlu Ile Arg Ser Phe Ala Glu 195 200 205 aaa gaa ttt tca gat aag ttt agagag gtt caa gca gga gaa cta aaa 672 Lys Glu Phe Ser Asp Lys Phe Arg GluVal Gln Ala Gly Glu Leu Lys 210 215 220 cac cat cta ttc cct ccc tgt gttaaa aat gct ctc aga gga gtt cca 720 His His Leu Phe Pro Pro Cys Val LysAsn Ala Leu Arg Gly Val Pro 225 230 235 240 cag gga atg agg aac tat gcaata acg gta ttg ctc acg agc ttt cta 768 Gln Gly Met Arg Asn Tyr Ala IleThr Val Leu Leu Thr Ser Phe Leu 245 250 255 agc tat gca agg ata tgt ccaaat cct ccc agg aga aat gta aaa att 816 Ser Tyr Ala Arg Ile Cys Pro AsnPro Pro Arg Arg Asn Val Lys Ile 260 265 270 agg gac tgc att aaa gat atgagg gta ata acc gag gaa ata ctt ccc 864 Arg Asp Cys Ile Lys Asp Met ArgVal Ile Thr Glu Glu Ile Leu Pro 275 280 285 ata ata ata gag gcc ggg aacaga tgc tca cct cca cta ttc gaa gat 912 Ile Ile Ile Glu Ala Gly Asn ArgCys Ser Pro Pro Leu Phe Glu Asp 290 295 300 caa cca aac gaa ata aag aatata tgg tac cac ttg ggc ttt gga tac 960 Gln Pro Asn Glu Ile Lys Asn IleTrp Tyr His Leu Gly Phe Gly Tyr 305 310 315 320 act gca aat cct acc cttgaa gac agc ggg aac tca aca tgg tac ttt 1008 Thr Ala Asn Pro Thr Leu GluAsp Ser Gly Asn Ser Thr Trp Tyr Phe 325 330 335 ccc cct aac tgt gat aagata aag gca aat gct cca cag ctt tgc act 1056 Pro Pro Asn Cys Asp Lys IleLys Ala Asn Ala Pro Gln Leu Cys Thr 340 345 350 cct gac aag cac tgc agatac att aga aat ccc cta aca tat tat cta 1104 Pro Asp Lys His Cys Arg TyrIle Arg Asn Pro Leu Thr Tyr Tyr Leu 355 360 365 agg cgt ctt tac tta gaagag aag agg agg gcc aag cat gct gat gag 1152 Arg Arg Leu Tyr Leu Glu GluLys Arg Arg Ala Lys His Ala Asp Glu 370 375 380 gga agt gac aaa gga ggaaag gag cga att cta cag taa 1191 Gly Ser Asp Lys Gly Gly Lys Glu Arg IleLeu Gln 385 390 395 5 30 DNA Artificial Sequence PCR primer 5 catatgctgatgagggaagt gacaaaggag 30 6 31 DNA Artificial Sequence PCR primer 6ctcgagcctt tattcatatt ccaaggactc t 31 7 28 DNA Artificial Sequence PCRprimer 7 cacgaccatg gtagacccat ttagtgag 28 8 30 DNA Artificial SequencePCR primer 8 cacggtcgac tcattactgt agaattcgct 30 9 49 DNA ArtificialSequence Probe 9 agctaccatg cctgcacgaa ttaagcaatt cgtaatcatg gtcatagct49

1. A protein having DNA primase activity and DNA polymerase activity. 2.The protein according to claim 1, wherein the protein is derived fromarchaebacteria.
 3. The protein according to claim 1, wherein the proteinhas thermal resistance.
 4. The protein according to any one of claims 1to 3, wherein the protein includes amino acid sequence shown in SEQ IDNO:
 1. 5. The protein according to any one of claims 1 to 3, wherein theprotein includes amino acid sequence wherein one or several amino acidsare deleted, replaced or added in the amino acid sequence shown in SEQID NO: 1
 6. A protein that enforces DNA primase activity and/or DNApolymerase activity by forming a complex with the protein according toany one of claims 1 to
 5. 7. The protein according to claim 6, whereinthe protein includes amino acid sequence shown in SEQ ID NO:
 2. 8. Theprotein according to claim 6, wherein the protein includes amino acidsequence wherein one or several amino acids are deleted, replaced oradded in amino acid sequence shown in SEQ ID NO:
 2. 9. A method ofproducing the protein according to claim 4 comprising the steps of: 1)preparing a base sequence that codes the protein having an amino acidsequence shown in SEQ ID NO: 1; 2) inserting the base sequence to anexpression vector; 3) transforming a host cell by the vector; 4)cultivating the transformant; and 5) isolating the protein described inclaim 4 from the culture.
 10. A method of producing the proteinaccording to claim 7 comprising the steps of: 1) preparing a basesequence that codes the protein having an amino acid sequence shown inSEQ ID NO: 2; 2) inserting the base sequence to an expression vector; 3)transforming a host cell by the vector; 4) cultivating the transformant;and 5) isolating the protein described in claim 7 from the culture. 11.A method of polymerizing deoxynucleotide triphosphate comprising thesteps of: 1) providing DNA chain as template DNA and deoxynucleotidetriphosphate as substrate; and 2) adding a protein that is described inany one of claims 1 to 5 and polymerize deoxynucleotide triphosphate.12. The method according to claim 11, further comprising the step ofadding a protein described in any one of claims 6 to
 8. 13. A method ofpolymerizing deoxynucleotide triphosphate comprising the steps of: 1)providing DNA chain as template DNA and deoxynucleotide triphosphate assubstrate; 2) adding complementary oligodeoxynuclotide to the DNA chainas template DNA and form complex with the DNA chain; and 3) adding aprotein that is described in any one of claims 1 to 5 and polymerizedeoxynucleotide triphosphate.
 14. The method according to claim 13,further comprising the step of adding a protein described in any one ofclaims 6 to 8.